Recombinant super-compound interferon

ABSTRACT

This invention provides a recombinant super-compound interferon or an equivalent thereof with changed spatial configuration. The super-compound interferon possesses anti-viral or anti-tumor activity and, therefore, is useful in preventing and treating viral diseases and cancers. This invention also provides an artificial gene which codes for the super-compound interferon or its equivalent. Finally, this invention provides methods for producing recombinant super-compound interferon or its equivalent and various uses of said interferon.

The application disclosed herein claims benefit of U.S. Ser. No.60/498,449, Filed Aug. 28, 2003. This application claims priority ofIndian Application No. 279/MUM/2004, filed Mar. 5, 2004, and IndianApplication No. 280/MUM/2004, filed Mar. 5, 2004. The contents of thepreceding applications are hereby incorporated in their entireties byreference into this application.

Throughout this application, various publications are referenced.Disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

This invention is related to a recombinant super-compound interferon(rSIFN-co) with changed spatial configuration. One characteristic ofrSIFN-co in this invention is that it inhibits DNA (deoxyribonucleicacid) duplication of the hepatitis B virus as well as the secretion ofHBsAg and HBeAg.

BACKGROUND OF THE INVENTION

rSIFN-co is a new interferon molecule constructed with the most popularconservative amino acid found in natural human α-IFN subtypes usinggenetic engineering methods. U.S. Pat. Nos. 4,695,623 and 4,897,471 havedescribed it. rSIFN-co had been proved to have broad-spectrum IFNactivity and virus- and tumor-inhibition and natural killer cellactivity. U.S. Pat. No. 5,372,808 by Amgen, Inc. addresses treatmentrSIFN-co. Chinese Patent No. 97193506.8 by Amgen, Inc. addressesre-treatment of rSIFN-co on hepatitis C. Chinese Patent No. 98114663.5by Shenzhen Jiusheng Bio-engineering Ltd. addresses treatment ofrSIFN-co on hepatitis B and hepatitis C.

The United States Food and Drug Administration (FDA) authorized Amgen,Inc. to produce rSIFN-co with E. Coli. for clinical hepatitis Ctreatment at the end of 1997.

Hepatitis B patients can be identified when detecting HBsAg and HBeAg.α-IFN is commonly used in clinics to treat hepatitis B. IFN bindssuperficial cell membrane receptors, inhibiting DNA and RNA (ribonucleicacid) duplication, including inducing some enzymes to preventduplication of the virus in hepatitis-infected cells. All IFNs caninhibit only the DNA duplication of viruses, not the e and s antigen.

This disclosure describes recombinant super-compound interferon, methodto produce the same, and uses thereof.

SUMMARY OF THE INVENTION

This invention provides a recombinant super-compound interferon or anequivalent thereof with changed spatial configuration. An equivalent isa molecule which is similar in function to the super-compoundinterferon. The super-compound interferon possesses anti-viral oranti-tumor activity. This invention also provides artificial gene whichcodes for the super-compound interferon or its equivalent.

This invention provides a process for production of recombinantsuper-compound interferon comprising introducing an artificial gene withselected codon preference into an appropriate host, culturing saidintroduced host in an appropriate condition permitting expression ofsaid super-compound interferon, and harvesting the expressedsuper-compound interferon.

This invention provides a composition comprising the recombinantsuper-compound interferon or its equivalent and a suitable carrier. Thisinvention further provides a pharmaceutical composition comprising therecombinant super-compound interferon or its equivalent and apharmaceutically acceptable carrier.

This invention provides a method for treating viral diseases or tumor ina subject comprising administering to the subject an effective amount ofthe super-compound interferon or its equivalent.

This invention provides the above-described method whereinsuper-compound interferon was administered orally, via vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal ormucosal administration, or by inhalation via an inspirator.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. rSIFN-co cDNA sequence designed according to E. Coli. codonusage and deduced rSIFN-co amino acid sequence

FIG. 2. Sequence of another super-compound interferon

FIG. 3. Diagram of pLac T7 cloning vector plasmid

FIG. 4. Diagram of pHY-4 expression vector plasmid

FIG. 5. Construction process of expression plasmid pHY-5

FIG. 6-A. Circular Dichroism spectrum of Infergen®

(Tested by Analysis and Measurement Center of Sichuan University)

Spectrum range: 250 nm-190 nm

Sensitivity: 2 m°/cm

Light path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml IFN-con1, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

Infergen® (interferon alfacon-1), made by Amgen Inc., also known asconsensus interferon, is marketed for the treatment of adults withchronic hepatitis C virus (HCV) infections. It is currently the only FDAapproved, bio-optimized interferon developed through rational drugdesign and the only interferon with data in the label specifically fornon-responding or refractory patients. InterMune's sales forcere-launched Infergen® in January 2002 with an active campaign to educateU.S. hepatologists about the safe and appropriate use of Infergen®,which represents new hope for the more than 50 percent of HCV patientswho fail other currently available therapies. Seehttp://www.intermune.com/wt/itmn/infergen, Aug. 27, 2003

FIG. 6-B. Circular Dichroism spectrum of Infergen® From Reference[Journal of Interferon and Cytokine Research. 16:489-499(1996)]

Circular dichroism spectra of concensus interferon subforms. Concensusinterferon was fractionated using an anion exchange column. Samples weredialyzed into 10 mM sodium phosphate, pH 7.4. Measurements were made onJasco J-170 spectopolarimeter, in a cell thermostat at 15° C. (-),acylated form; ( - - - ) cis terminal form; ( . . . ), met terminalform. A. Far UV Spectrum. B. Near UV Spectrum.

FIG. 6-C. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 320 nm-250 nm

Sensitivity: 2 m°/cm

Light path: 2 cm

Equipment: Circular Dichroism J-500C

Samples: contains 0.5 mg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

FIG. 6-D. Circular Dichroism spectrum of rSIFN-co

Spectrum range: 250 nm-190 nm

Sensitivity: 2 m°/cm

Light path: 0.20 cm

Equipment: Circular Dichroism J-500C

Samples: contains 30 μg/ml rSIFN-co, 5.9 mg/ml of NaCl and 3.8 mg/ml ofNa₂PO₄, pH7.0.

Clearly, as evidenced by the above spectra, the secondary or eventertiary structure of rSIFN-co is different from Infergen®.

FIG. 7. Comparison of Inhibition Effects of Different Interferons on HBVGene Expression

FIG. 8A-1. Curves of Changes of Body Temperature in Group A (5 patients)

This figure is the record of body temperature changes of 5 patients inGroup A.

FIG. 8A-2. Curves of Changes of Body Temperature in Group A (6 patients)

This figure is the record of body temperature changes of the other 6patients in Group A.

FIG. 8B-1. Curves of Changes of Body Temperature in Group B (5 patients)

This figure is the record of body temperature changes of 5 patients inGroup B.

FIG. 8B-2. Curves of Changes of Body Temperature in Group B (5 patients)

This figure is the record of body temperature changes of the other 5patients in Group B.

FIG. 9. rsIFN-co Crystal I

FIG. 10. rsIFN-co Crystal II

FIG. 11. The X-ray Diffraction of rsIFN-co Crystal

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for producing a recombinantsuper-compound interferon with changed spatial configuration andenhanced antiviral activity comprising steps of:

-   -   (a) Introducing nucleic acid molecule which codes for said        interferon with preferred codons for expression to an        appropriate host; and    -   (b) Placing the introduced host in conditions allowing        expression of said interferon.

This invention provides the method for producing interferon, furthercomprising recovery of the expressed interferon.

This invention provides a recombinant super-compound interferon or anequivalent thereof with changed spatial configuration. This inventionreveals that proteins with the same primary sequence might havedifferent biological activities. As illustrated in the followingexample, this invention discloses two proteins with identical amino acidsequences but with different activities. The efficacy of this activitymay sometimes be improved and, sometimes, the protein with changedspatial configuration would reveal new function.

An equivalent is a molecule which is similar in function to the compoundinterferon. An equivalent could be a deletion, substitution, orreplacement mutant of the original sequence. Alternatively, it is alsothe intention of this invention to cover mimics of the recombinantsuper-compound interferon. Mimics could be a peptide, polypeptide or asmall chemical entity.

The interferon described herein includes but is not limited tointerferon α, β, or ω. In an embodiment, it is IFN-1a, IFN-2b or othermutants.

In an embodiment, the super-compound interferon disclosed has higherefficacy than the interferon described in U.S. Pat. Nos. 4,695,623 or4,897,471. This super-compound interferon is believed to have uniquesecondary or tertiary structure. (See e.g. FIG. 6.)

The super-compound interferon described herein has spatial structurechange(s) resulting from the changes of its production process.

The above-described super-compound interferon may be produced by ahigh-efficiency expression system which uses a special promoter. In anembodiment, the promoter is P_(BAD). As could be easily appreciated byother ordinary skilled artisans. Other inducible promoters, such as heatshock promoters or heavy metal inducible promoters, may be used in thisinvention.

The super-compound interferon may also be produced with its gene asartificially synthesized cDNA with adjustment of its sequence from thewild-type according to codon preference of E. Coli. Extensive discussionof said codon usage (preference) may be found in U.S. Pat. No.4,695,623. See e.g. column 6, line 41-column 7, line 35.

The above-described super-compound interferon possesses anti-viral oranti-tumor activity, and; therefore, is useful in inhibiting, preventingand treating viral diseases, tumors, or cancers.

As used herein, viral diseases include, but are not limited to,hepatitis A, hepatitis B, hepatitis C, other types of hepatitis,infections caused by Epstein-Barr virus, Cytomegalovirus, herpes simplexviruses, other herpes viruses, papovaviruses, poxviruses,picornaviruses, adenoviruses, rhinoviruses, human T-cell leukemia virusI, human T-cell leukemia virus II, or human T-cell leukemia virus III.

Viral upper respiratory infection, alternative names common cold, colds.This is a contagious viral infection of the upper respiratory tractcharacterized by inflammation of the mucous membranes, sneezing, and asore throat. It is usually caused by over 200 different viruses, knownas rhinoviruses. Colds are not caused by the same viruses responsiblefor influenza. Colds are spread through droplets from the coughing orsneezing of others with a cold or by hand contact with objectscontaminated by someone with a cold. The incidence of colds is highestamong children, and the incidence decreases with age because immunity tothe virus causing the cold occurs after the illness. Gradually, immunityto a wide variety of viruses that cause colds is developed in adults.Children may have 10 colds a year, and adults may have 3 colds a year.

The U.S. Centers for Disease Control and Prevention have estimated thatthe average annual incidence of upper respiratory tract infections(URIs) in the United States is 429 million episodes, resulting in morethan $2.5 billion in direct and indirect healthcare costs.

The common cold is most often caused by one of several hundredrhinoviruses (52%), but coronaviruses (8%) or the respiratory syncytialvirus (7%) may also lead to infection. Other viruses, such as influenza(6%), parainfluenza, and adenoviruses, may produce respiratory symptoms,but these are often associated with pneumonia, fever, or chills.

Colds occur in a seasonal pattern that usually begins in mid-Septemberand concludes in late April to early May. The common cold is quitecontagious and can be transmitted by either person-to-person contact orairborne droplets. Upper respiratory symptoms usually begin 1 to 2 daysafter exposure and generally last 1 to 2 weeks, even though viralshedding and contagion can continue for 2 to 3 more weeks. Symptoms maypersist with the occurrence of complications such as sinusitis or lowerrespiratory involvement such as bronchitis or pneumonia.

The common cold has a variety of overt symptoms, including malaise,nasal stuffiness, rhinorrhea, nonproductive cough, mild sore throat,and, in some cases, a low-grade fever. Because of the similarity ofsymptoms, a cold may be mistaken for perennial allergic rhinitis, butallergies can usually be ruled out because of the differences inchronicity.

If a patient presents with a viral URI, the spectrum of remedies isextensive. Since most of these infections are self-limiting, cliniciansusually recommend rest and fluids, but other treatments includeenvironmental and nutritional therapies, over-the-counter andprescription decongestant and antihistamine products, new antihistamineand anticholinergic nasal formulations, and antibiotics. Table 1 listscommonly used cough and cold medications and their side effects.

TABLE 1 A Profile of Common Cough and Cold Medications and their sideeffects Side Effects and Special Medication Purpose ConsiderationsAerosolized beta2 Reverse Raises heart rate and may agonists (eg,postinflammatory cause tremor albuterol) bronchospasm Alcohol-basedliquid Treat multiple Potential drowsiness and combination productssymptoms coordination problems Alpha1 agonists Decongestion May causetachycardia, (oral) (eg, nervousness, transient pseudoephedrine,stimulation, dizziness, phenylpropanolamine) drowsiness, elevation ofblood pressure Anticholinergic Drying May cause nasal dryness compounds:and occasional epistaxis Ipratropium bromide (topical) Other Drying Maycause orthostasis, anticholinergics dysfunction of heat (eg, regulation,dry mouth, methscopolamine, constipation atropine, hyoscyamine)Antihistamines Drying Drowsiness, dry mouth, (oral) (eg, orthostatichypertension chlorpheniramine, diphenhydramine) Benzonatate capsulesCough suppression, Chewing can numb local anesthesia the mouth; cancause sedation, dizziness Codeine, hydrocodone Cough suppressionDrowsiness, constipation, nausea Dextromethorphan Cough suppressionDrowsiness possible, but side effects uncommon Guaifenesin Promote Noside effects; must be expectoration taken with lots of water to(mucolysis) improve efficacy Topical Decongestion Local burning;prolonged decongestants (eg, use may cause dependence oxymetazoline,phenylephrine) Zinc and vitamin C Possible reduction Possible tastedisturbance, lozenges in symptom severity increase of oxalate stones andduration if susceptible

Abstract from http://www.physsportsmed.com/issues/1998/02feb/swain.htm

Usage of Super-Compound Interferon to Prevent or Treat URI

Nearly 70˜80% URI are caused by viruses such as respiratory Syncyticalvirus, adenovirus, rhinovirous, cox-sackie virus, corona virus and itsvariant, influenza A virus and its variant, influenza B virus and itsvariant, parainfluenza virus and its variant, or enterovirus and itsvariant. A main cause of URI in adults is from rhinovirous. Forchildren, respiratory syncytical virus and parainfluenza virus are twoleading causes of URI.

Super-compound interferon plays an important role in the fight againstviruses that cause URI. Super-compound interferon gains its anti-virusaffects mainly via two mechanisms:

-   -   1. Attach to surface of sensitive cells and induce them to        product anti-virus protein, then block the duplication and        reproduction of viruses in vivo.    -   2. Super-compound interferon can adjust immune response,        including T-cell immune response, activity of NK cell, the        phagocytosis function of monokaryon, and even formation of some        antibodies in vivo.

In treatment for URI, Super-compound interferon can be directly appliedto the affected area via a spray inspiration. This method of treatmentallows the interferon to reach the target cells first hand.Consequently, marketing the supply as a spray, rather than via oral orinjection, would be safer and more effective for administrating theinterferon.

Usage of Super-Compound Interferon to Prevent or Treat SARS

With the consent of the Sichuan working group on SARS prevention andcontrol, the distribution of Super-compound interferon began in May of2003. Super-compound interferon spray was allocated to doctors andnurses in hospitals, populated areas with a high risk for SARS, and tothe National research group on prevention and control of SARS. Among the3,000 users as of Dec. 19, 2003, there were no reports of any sideeffects connected to the use of the spray. Furthermore, none of thedoctors and nurses, the people of Sichuan Province, or otherorganizations that have used the Super-compound interferon spray hasbeen infected by SARS.

Therefore, this invention provides a method for inhibiting, preventingor treating virus replication or virus-infected cells by contacting saidvirus or infected cells with an effective amount of the super-compoundinterferon or its equivalent.

This super-compound interferon is useful in inhibiting, preventing ortreating the following cancers or tumors:

Cancer Skin Cancer Basal Cell Carcinoma Malignant Melanoma Renal cellcarcinoma Liver Cancer Thyroid Cancer Rhinopharyngeal Cancer SolidCarcinoma Prostate Cancer Stomach/Abdominal Cancer Esophageal CancerRectal Cancer Pancreatic Cancer Breast Cancer Ovarian Cancer &Superficial Bladder Cancer Hemangioma Epidermoid Carcinoma CervicalCancer Non-small Cell Lung Cancer Small Cell Lung Cancer GliomaMalignant Leucocythemia Acute Leucocythemia Hemal Chronic LeucocythemiaDisease Chronic Myelocytic Leukemia Hairy Cell Leukemia LymphadenomaMultiple Myeloma Polycythemia Vera Others Kaposi's Sarcoma

Patient #1. A female patient with ovarian cancer started receivinginjections. She received 15 μg injections on July 14^(th), July 16^(th),July 18^(th), July 20^(th), and July 22^(nd). On July 14^(th), 2000 mlof peritoneal fluid was observed. The patient underwent chemotherapy onJuly 22^(nd). On August 3^(rd), the patient's peritoneum was opened. 21of fluid was expected to be found, but only 200 ml of fluid wasobserved. The left and right ovaries and lymphatic nodes were cancerous.All other organs were clear.

Patient #2. A kidney cancer patient was treated in the following manner.In a half-month period, the patient was given 3 injections of 9 μg ofrSIFN-co and 3 injections of 15 μg of rSIFN-co. In the one full monthfollowing these injections, he received 9 μg and 15 μg injections ofrSIFN-co every other day. A kidney biopsy showed no metastasis afterthis course of treatment. The patient showed a full recovery. Every halfyear after recovery, the patient received 15 μg injections of rSIFN-co15 times over a one-month period.

Accordingly, this invention provides a method for inhibiting tumor orcancer cell growth by contacting the super-compound interferon or itsequivalent with said tumor or cancer cells.

In a further embodiment, the super-compound interferon inhibits the DNAduplication and secretion of HBsAg and HBeAg of Hepatitis B Virus.

This invention also provides an artificial gene which codes for thesuper-compound interferon or its equivalent. It is within the ordinaryskill to design an artificial gene. Many methods for generatingnucleotide sequence and other molecular biology techniques have beendescribed previously. See for example, Joseph Sambrook and David W.Russell, Molecular Cloning: A laboratory Manual, December 2000,published by Cold Spring Harbor Laboratory Press.

This invention provides a vector comprising the gene which codes for thesuper-compound interferon or its equivalent.

This invention provides an expression system comprising the vectorcomprising the gene which codes for the super-compound interferon or itsequivalent. The cells include, but are not limited to, prokaryotic oreukaryotic cells.

This invention also provides a host cell comprising the vectorcomprising the gene which codes for the super-compound interferon or itsequivalent.

This invention provides a process for production of recombinantsuper-compound interferon comprising introducing an artificial gene withselected codon preference into an appropriate host, culturing saidintroduced host in an appropriate condition for the expression of saidcompound interferon and harvesting the expressed compound interferon.

The process may comprise extraction of super-compound interferon fromfermentation broth, collection of inclusion body, denaturation andrenaturation of the harvested protein.

The process may maintain the high efficacy even when the super-compoundinterferon is used with an agent and in a particular concentration. Theprocess also comprises separation and purification of the super-compoundinterferon. The process further comprises lyophilization of the purifiedsuper-compound interferon. The process comprises production of liquidinjection of super-compound interferon.

This invention also provides the produced super-compound interferon bythe above processes.

This invention provides a composition comprising the recombinantsuper-compound interferon or its equivalent and a suitable carrier.

This invention provides a pharmaceutical composition comprising therecombinant super-compound interferon or its equivalent and apharmaceutically acceptable carrier.

This invention provides a method for treating or preventing viraldiseases or tumors in a subject comprising administering to the subjectan effective amount of the super-compound interferon or its equivalent.

This invention provides the above-described method wherein the viraldiseases include, but are not limited to, hepatitis A, hepatitis B,hepatitis C, other types of hepatitis, infections of viruses caused byEpstein-Barr virus, Cytomegalovirus, herpes simplex viruses, or othertype of herpes viruses, papovaviruses, poxviruses, picornaviruses,adenoviruses, rhinoviruses, human T-cell leukemia viruses I, or humanT-cell leukemia viruses II, or human T-cell leukemia virus III.

This invention provides the above-described method whereinsuper-compound interferon was administered orally, via vein injection,muscle injection, peritoneal injection, subcutaneous injection, nasal ormucosal administration, or by inhalation via an inspirator.

This invention provides the above-described method whereinsuper-compound interferon was administered following the protocol ofinjection: 9 μg or 15 μg every two days, 3 times a week, for 24 weeks.

It was surprising to find that rSIFN-co, the spatial structure of whichhas been changed, is not only a preparation to inhibit the DNAduplication of hepatitis B, but to inhibit the secretion of HBsAg andHBeAg on 2.2.15 cells.

One objective of this invention is to offer a preparation of rSIFN-co todirectly inhibit the DNA duplication of hepatitis B viruses and thesecretion of HBeAg and HBsAg of hepatitis B and decrease them to normallevels.

In one embodiment, rSIFN-co was produced with recombinant techniques. Onthe condition of fixed amino acid sequence, the IFN DNA was redesignedaccording to the E. Coli. codon usage and then the rSIFN-co gene wasartificially synthesized. rSIFN-co cDNA was cloned into thehigh-expression vector of E. Coli. by DNA recombinant techniques, and ahigh expression of rSIFN-co was gained by using ofinduce/activate-mechanism of L-arabinose to activate the transcriptionof P_(BAD) promoter.

Compared with usual thermo-induction, pH induction and IPTG inductionsystems of genetic engineering, arabinose induction/activation systemhas some advantages: (1) Common systems relieve promoter function bycreating a “derepression” pattern. Promoters then induce downstream geneexpression. So temperature and pH change and the addition of IPTG cannotactivate promoters directly. In the system disclosed herein, L-arabinosenot only deactivates and represses but also activates the transcriptionof P_(BAD) promoter which induces a high expression of rSIFN-co.Therefore, the arabinose induction/activation system is a more effectiveexpression system. (2) The relationship between Exogenous andL-arabinose dosage is linear. This means the concentration of arabinosecan be changed to adjust the expression level of the exogenous gene.Therefore, it is easier to control the exogenous gene expression levelin E. Coli. by arabinose than by changing temperature and pH value. Thischaracteristic is significant for the formation of inclusion bodies. (3)L-arabinose is resourceful, cheap and safe, which, on the contrary, arethe disadvantages of other inducers such as IPTG.

This embodiment creates an effective and resistant rSIFN-co-expressingE. Coli. engineering strain with an L-arabinose induction/activationsystem. The strain is cultivated and fermented under suitable conditionsto harvest the bacterial bodies. Inclusion bodies are then purifiedafter destroying bacteria and washing repeatedly. The end result, massof high-purity, spatial-configuration-changed rSIFN-co protein for thisinvention and for clinical treatment, was gained from denaturation andrenaturation of inclusion bodies and a series of purification steps.

The following are some rSIFN-co preparations: tablets, capsules, liquidsfor oral consumption, pastes, injections, sprays, suppositories, andsolutions. Injections are recommended. It is common to subcutaneouslyinject or vein-inject the medicine. The medicine carrier could be anyacceptable medicine carrier, including carbohydrates, cellulosum,adhesive, collapse, emollient, filling, add-dissolving agent,amortization, preservative, thickening agent, matching, etc.

This invention also provides a pharmaceutical composition comprising theabove composition and a pharmaceutically acceptable carrier.

For the purposes of this invention, “pharmaceutically acceptablecarriers” means any of the standard pharmaceutical carriers. Examples ofsuitable carriers are well known in the art and may include, but are notlimited to, any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution and various wetting agents. Othercarriers may include additives used in tablets, granules, capsules, etc.Typically such carriers contain excipients such as starch, milk, sugar,certain types of clay, gelatin, stearic acid or salts thereof, magnesiumor calcium stearate, talc, vegetable fats or oils, gum, glycols or otherknown excipients. Such carriers may also include flavor and coloradditives or other ingredients. Compositions comprising such carriersare formulated by well-known conventional methods.

This invention provides a method for preventing or treating Severe AcuteRespiratory Syndrome, or virus-induced upper respiratory diseases, of asubject comprising administering to the subject an effective amount ofrecombinant super-compound interferon or a functional equivalentthereof.

In an embodiment of the above method, the interferon is α, β, or ω.

The super-compound interferon may be administered orally, via veininjection, muscle injection, peritoneal injection, subcutaneousinjection, nasal or mucosal administration, or by inhalation via aninspirator.

In an embodiment, the interferon is delivered by a spray device.

In a specific embodiment, the device is described in FIG. 7.

In one of the embodiments, the interferon is lyophilized.

This invention provides a method for inhibiting the causative agent ofSevere Acute Respiratory Syndrome, or virus-induced upper respiratorydiseases, comprising contacting the agent with an effective amount ofsuper-compound interferon or its equivalent.

It is determined that the causative agent of SARS is a virus. See eg.Rota et al (2003), Characterization of a Novel Coronavirus Associatedwith Severe Acute Respiratory Syndrome. Science 1085952www.sciencexpress.org and Marra, et al. (2003), The Genome Sequence ofthe SARS-Associated Coronavirus. Science 1085853 www.sciencexpress.org.

This invention also provides a method for inhibiting Severe AcuteRespiratory Syndrome virus or Severe Acute Respiratory Syndromevirus-infected cells, or virus-induced upper respiratory diseases, orcells infected with viruses capable of inducing upper respiratorydiseases, comprising contacting an effective amount of thesuper-compound interferon with said virus or cell. This contact could bedirect or indirect.

This invention provides a composition comprising an effective amount ofthe super-compound interferon capable of inhibiting Severe AcuteRespiratory Syndrome virus or Severe Acute Respiratory Syndromevirus-infected cells, or virus-induced upper respiratory diseases, orcells infected with viruses capable of inducing upper respiratorydiseases, and a suitable carrier.

This invention provides a composition comprising an effective amount ofthe super-compound interferon capable of preventing or treating SevereAcute Respiratory Syndrome, or virus-induced upper respiratory diseases,of a subject and a suitable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the recombinant super-compound interferon capable ofinhibiting Severe Acute Respiratory Syndrome virus or Severe AcuteRespiratory Syndrome virus-infected cells, or virus-induced upperrespiratory diseases, and a pharmaceutically acceptable carrier.

This invention provides a pharmaceutical composition comprising aneffective amount of the recombinant super-compound interferon capable ofpreventing or treating Severe Acute Respiratory Syndrome, orvirus-induced upper respiratory diseases, in a subject and apharmaceutically acceptable carrier.

This invention provides a device to deliver the above-describedpharmaceutical composition.

In a preferred embodiment, the subject is a human. As it can easily beappreciated, the super-compound interferon can be used in other animalsor mammals.

This invention provides a method for preventing Severe Acute RespiratorySyndrome or virus-induced upper respiratory diseases, in humanscomprising application of the super-compound interferon three times aday via a spray which contains twenty micrograms of interferon, equal toten million units of activity in three milliliter.

This invention will be better understood from the examples which follow.However, one skilled in the art will readily appreciate that thespecific methods and results discussed are merely illustrative of theinvention as described more fully in the claims which follow thereafter.

Experimental Details Example 1

rSIFN-co is a new interferon molecule constructed according toconservative amino acid in human IFN-α subtype with genetic engineeringmethod. It has been proven that rSIFN-co has broad-spectrum IFNactivity, such as high antivirus and tumor inhibition activity,especially for effectively treating hepatitis C.

E. Coli. codon was used to redesign rSIFN-co cDNA and then artificiallysynthesize cDNA of rSIFN-co from published rSIFN-co DNA sequences anddeduced amino acid sequences (FIG. 1).

In order to get pure rSIFN-co protein, rSIFN-co cDNA was cloned into E.Coli. high-expression vector, and L-arabinose, which can activate strongP_(BAD) promoter in vectors, was used to induce high expression ofrSlFN-co gene.

Synthesis of E. Coli. cDNA Sequence

Redesign of rSIFN-co cDNA Sequence

rSIFN-co cDNA was redesigned according to the codon usage of E. Coli. toachieve high expression in E. Coli. Deduced amino acid sequence from theredesigned cDNA sequence of rSIFN-co is completely coincidental withprimitive amino acid sequence of published rSIFN-co (FIG. 1).

rSIFN-co cDNA Sequence Synthesis

rSIFN-co cDNA 5′-Terminus and 3′-Terminus Semi-Molecular Synthesis

Two semi-moleculars can be directly synthesized: rSIFN-co cDNA5′-terminus 280 bp (fragment I) and 3′-terminus 268 bp (fragment II) byPCR. There are 41 bp overlapping among fragment II and fragment I.

(1) Chemical Synthesis Oligodeoxynucleotide Fragment:

Oligomer A: 5′ATGTGCGACCTGCCGCAGACCCACTCCCTGGGTAACCGTCGTGCTCTGATCCTGCTGGCTCAGATGCGTCGTATCTCCCCGTTCTCCTGCCTGAAAGA CCGTCACGAC3′ OligomerB: 5′CTGAAAGACCGTCACGACTTCGGTTTCCCGCAGGAGAGGTTCGACGGTAACCAGTTCCAGAAGCTCAGGCTATCTCCGTTCTGCACGAAATGATCCAG CAGACCTTC3′ OligomerC: 5′GCTGCTGGTACAGTTCGGTGTAGAATTTTTCCAGCAGGGATTCGTCCCAAGCAGCGGAGGAGTCTTTGGTGGAGAACAGGTTGAAGGTCTGCTGGATC ATTTC3′ Oligomer D:5′ATCCCTGCTGGAAAAATTCTACACCGAACTGTACCAGCAGCTGAACGACCTGGAAGCTTGCGTTATCCAGGAAGTTGGTGTTGAAGAAACCCCGCTGA TGAAC3′ Oligomer B:5′GAAGAAACCCCGCTGATGAACGTTGACTCCATCCTGGCTGTTAAAAAATACTTCCAGCGTATCACCCTGTACCTGACCGAAAAAAAATACTCCCCGTG CGCTTGGG3′ OligomerF: 5′TTATTCTTTACGACGCAGACGTTCCTGCAGGTTGGTGGACAGGGAGAAGGAACGCATGATTTCAGCACGAACAACTTCCCAAGCGCACGGGGAGTATT TTTTTTCGGTCAGG3′

PCR I for Fragment I: oligodeoxynucleotide B as template,oligodeoxynucleotide A and C as primers, synthesized 280 by Fragment I.

PCR I mixture (units: μl) sterilized distilled water 39 10 × Pfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 Oligomer A primer (25 μmol/L) 1 Oligomer C primer (25 μmol/L) 1Oligomer B template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen AmericanLtd.) (25 U/μl) 1 Total volume 50 μl PCR cycle: 95 I 2 m→ (95° C.45s→65° C.1 m→72° C.1 m) × 25 cycle→72° C.10 m→′4° C.

-   -   PCR II for Fragment II: oligodeoxynucleotide E as template,        oligodeoxynucleotide D and F as primers, synthesized 268 bp        Fragment II.

PCR II mixture (units: μl) sterilized distilled water 39 10 × Pfu buffer(Stratagen American Ltd.) 5 dNTP mixture (dNTP concentration 2.5 mmol/L)2 Oligomer D primer (25 μmol/L) 1 Oligomer F primer (25 μmol/L) 1Oligomer E template (1 μmol/L) 1 Pfu DNA polymerase (Stratagen AmericanLtd.) 1 (25 U/μl) Total volume 50 μl PCR cycle: the same as PCR I

Assembling of rSIFN-co cDNA

Fragment I and II were assembled together to get the complete cDNAmolecular sequence of rSIFN-co using the overlapping and extending PCRmethod. Restriction enzyme Nde I and Pst I were introduced to clonerSIFN-co cDNA sequence into plasmid.

-   -   (1) Chemical synthesis primers

Oligomer G: 5′ATCGGCCATATGTGCGACCTGCCGCAGACCC3′ Oligomer H:5′ACTGCCAGGCTGCAGTTATTCTTTACGACGCAGACGTTCC3′

-   -   (2) Overlapping and extending PCR

PCR mixture (units: μl) sterilized distilled water 38 10 × Pfu buffer(Stratagen American Ltd.)  5 dNTP mixture (dNTP concentration 2.5mmol/L)  2 primer G (25 μmol/L)  1 primer H (25 μmol/L)  1 *fragment Ipreduction (1 μmol/L)  1 *fragment II preduction (1 μmol/L)  1 Pfu DNApolymerase (Stratagen American Ltd.) (2.5 U/μl)  1 Total volume 50μ*Separate and purify PCR production with StrataPrep PCR purification kitproduced by Stratagen American Ltd. And dissolve into sterilizeddistilled water. PCR cycle: the same as PCR I

rSIFN-co Gene Clone and Sequence Analysis

pLac T7 plasmid as cloning vector. pLac T7 plasmid is reconstructed withpBluescript II KS(+) plasmid produced by Stratagen (FIG. 3).

Purified PCR production of rSIFN-co cDNA with StrataPrep PCRpurification kit. Digest cDNA and pLac T7 plasmid with NdeI and PstI.Run 1% agarose gel electrophoresis and separate these double-digestedDNA fragments. Recover 507 bp long rSIFN-co DNA fragment and 2.9 kbplasmid DNA fragment. Ligate these fragments by T4 DNA ligase to form arecombinant plasmid. Transform DH_(5α)competent cells (Gibco) with therecombinant plasmid, culture at 37° C. overnight. Identify the positiverecombinant colony, named pHY-1.

Run DNA sequencing with SequiTherm™ Cycle Sequencing Kit produced byAmerican Epicentre Technologies Ltd using L1-COR Model 4000L. Primersare T7 and T3 common sequence primer, the DNA sequencing result matchestheoretic design.

Purify the rSIFN-co, sequence the N-terminus amino acids, the N-terminusamino acid sequence matches experimental design which is as follows:

N- Cys-Asp-Leu-Pro-Gln-Thr-His-Ser-Leu-Gly-Asn- Arg-Arg-Ala-Leu

Construction, Transformation, Identification, and Hereditary Stabilityof Expression Vector

Construction and Transformation of Expression Vector

Digested E. Coli. expression vector pHY-4 (see FIG. 3) with Nde I tolinearize and subsequently digest with Xba I. Run 1% agarose gelelectrophoresis, and purify the 4.8 kb pHY-4 Nde I-Xba I digest fragmentwith QIAEX II kit produced by QIAGEN Germany Ltd.

At the same time, the pHY-4 plasmid is double digested with Nde I-Xba I.Run 1% agarose gel electrophoresis and purify the 715 bp fragment.Ligate the rSIFN-co and pHY-4 fragments with T4 DNA ligase to constructthe recombinant plasmid (See FIG. 4). Transform DH_(5α)competent cellswith the recombinant plasmid. Spread the transformed cells on LB platewith Amp, 37° C. culture overnight.

Positive Cloning Strain Screening

Randomly choose E. Coli. colonies from above LB-plate, screening thepositive strains containing recombinant vector by endonuclease digestingand PCR analysis. Name one of the positive recombinant plasmid pHY-5,and name the strain containing pHY-5 plasmid PVIII. Amplify and storethe positive strain with glycerol in −80° C.

High Expression of rSIFN-co Gene in E. Coli.

In pHY-5 plasmid, rSIFN-co gene is under control of strong promoterP_(BAD). This promoter is positively and negatively regulated by theproduct of the gene araC. AraC is a transcriptional regulator that formsa complex with arabinose. In the absence of arabinose, the AraC dimerbinds O₂ and I₁ forming a 210 bp loop. This conformation leads to acomplete inhibition of transcription. In the presence of arabinose, thedimer is released from O₂ and binds I₁ and I₂ leading to transcription.Arabinose binding deactivates, represses and even activates thetranscription of P_(BAD) promoter, which stimulates P_(BAD) inducinghigh expression of rSIFN-co. rSIFN-co expression level in PVIII is morethan 50% of the total E. Coli. protein.

SUMMARY

RSIFN-CO is a new interferon molecule artificially built according tothe conservative amino acid of human α interferons. It has been provenas a effective anti-hepatitis drug. In order to get enough pure rSIFN-coprotein, a stable recombinant E. Coli. strain which high expressesrSIFN-co protein was constructed.

First, according to published rSIFN-co amino acid sequence, E. Coli.codon was used to synthesize whole cDNA of rSIFN-co. This DNA fragmentwas sequenced and proved that the 501 bp codon sequence and TAAtermination codon sequence are as expected and consistent with theexperimental design. Subsequent analysis revealed that the N-terminusamino acid sequence and amino acid composed of rSIFN-co produced by therecombinant strain were both consistent with the prediction.

The rSIFN-co cDNA was cloned into E. Coli. high-expression vector pHY-4plasmid to construct the recombinant plasmid pHY-5. E. Coli. LMG194strain was further transformed with pHY-4 plasmid to get stable rSIFN-cohigh-expression transformant. This transformant was cultured for 30generations. The heredity of pHY-5 recombinant plasmid in E. Coli.LMG194 was normal and stable, and the expression of rSIFN-co was highand steady.

E. Coli. LMG194, which contains recombinant pHY-5 plasmid, is actuallyan ideal high-expression engineering strain.

REFERENCES

1. Blatt L M, Davis J M, Klein S B. et al. The biologic activity andmolecular characterization of a novel synthetic interferon-alphaspecies, consensus interferon. Journal of Interferon and CytokineResearch, 1996;16(7):489-499.

2. Alton, K. et al: Production characterization and biological effectsof recombinant DNA derived human IFN-α and IFN-γ analogs. In: De MaegerE, Schellekens H. eds. The Biology of Interferon System. 2nd ed.Amsterdam: Elsevier Science Publishers, 1983: 119-128

3. Pfeffer L M. Biologic activity of natural and synthetic type 1interferons. Seminars in Oncology, 1997;24 (3 suppl 9):59-63-59-69.

4. Ozes O N, Reiter Z, Klein S, et al. A comparison of interferon-con1with natural recombinant interferons-(: antiviral, antiproliferative,and natural killer-inducing activities. J. Interferon Res., 1992;12:55-59.

5. Heathcote E J L, Keeffe E B, Lee S S, et al. Re-treatment of chronichepatitis C with consensus interferon. Hepatology, 1998;27(4):1136-1143.

6. Klein M L, Bartley T D, Lai P H, et al. Structural characterizationof recombinant consensus interferon-alpha. Journal of Chromatography,1988; 454:205-215.

7. The Wisconsin Package, by Genetics Computer Group, Inc. Copyright1992, Medison, Wis., USA

8. Nishimura, A et al: A rapid and highly efficient method forpreparation of competent E. coli cells. Nuclei. Acids Res. 1990, 18:6169

9. All molecular cloning techniques used are from: Sambrook, J., E. F.Fritsch and T. Maniatis. Molecular Cloning: A laboratory manual, 2nd ed.CSH Laboratory Press, Cold Spring Harbour, N.Y. 1989.

10. Guzman, L. M et al: Tight regulation, modulation, and high-levelexpress-ion by vectors containing the arabinose PBAD promoter. J.Bacteriol. 1995, 177: 4121˜4130.

rSIFN-co cDNA Sequence Designed According to E. COLI. Codon Usage andDeduced rSIFN-co Amino Acid Sequence

5′   11    21    31   41   51 +1 M C D L P Q T H S L G N R R A L I L L A1 ATGTGCGACC TGCCGCAGAC CCACTCCCTG GGTAACCGTC GTGCTCTGAT CCTGCTGGCTTACACGCTGG ACGGCGTCTG GGTGAGGGAC CCATTGGCAG CACGAGACTA GGACGACCGA5′   71    81    91   101   111 +1 Q M R R I S P F S C L K D R H D F G FP 61 CAGATGCGTC GTATCTCCCC GTTCTCCTGC CTGAAAGACC GTCACGACTT CGGTTTCCCGGTCTACGCAG CATAGAGGGG CAAGAGGACG GACTTTCTGG CAGTGCTGAA GCCAAAGGGC5′   131   141   151   161   171 +1 Q E E F D G N Q F Q K A Q A I S V LH E 121 CAGGAAGAAT TCGACGGTAA CCAGTTCCAG AAAGCTCAGG CTATCTCCGTTCTGCACGAA GTCCTTCTTA AGCTGCCATT GGTCAAGGTC TTTCGAGTCC GATAGAGGCAAGACGTGCTT 5′   191   201   211   221   231 +1 M I Q Q T F N L F S T K DS S A A W D E 181 ATGATCCAGC AGACCTTCAA CCTGTTCTCC ACCAAAGACT CCTCCGCTGCTTGGGACGAA TACTAGGTCG TCTGGAAGTT GGACAAGAGG TGGTTTCTGA GGAGGCGACGAACCCTGCTT 5′   251   261   271   281   291 +1 S L L E K F Y T F L Y Q QL N D L F A C 241 TCCCTGCTGG AAAAATTCTA CACCGAACTG TACCAGCAGC TGAACGACCTGGAAGCTTGC AGGGACGACC TTTTTAAGAT GTGGCTTGAC ATGGTCGTCG ACTTGCTGGACCTTCGAACG 5′      311   321      331   341   351 +1 V I Q E V G V E E TP L M N V D S I L A 301 GTTATCCAGG AAGTTGGTGT TGAAGAAACC CCGCTGATGAACGTTGACTC CATCCTGGCT CAATAGGTCC TTCAACCACA ACTTCTTTGG GGCGACTACTTGCAACTGAG GTAGGACCGA 5′     371   381    391   401   411 +1 V K K Y F QR I T L Y L T E K K Y S P C 361 GTTAAAAAAT ACTTCCAGCG TATCACCCTGTACCTGACCG AAAAAAAATA CTCCCCGTGC CAATTTTTTA TGAAGGTCGC ATAGTGGGACATGGACTGGC TTTTTTTTAT GAGGGGCACG 5′     431   441    451   461   471 +1A W E V V R A E I M R S F S L S T N L Q 421 GCTTGGGAAG TTGTTCGTGCTGAAATCATG CGTTCCTTCT CCCTGTCCAC CAACCTGCAG CGAACCCTTC AACAAGCACGACTTTAGTAC GCAAGGAAGA GGGACAGGTG GTTGGACGTC 5′    491    501 +1 E R L RR K E # 481 GAACGTCTGC GTCGTAAAGA ATAA CTTGCAGACG CAGCATTTCT TATT

Example 2 Separation and Purification of rSIFN-co

1. Fermentation

Inoculate the recombinant strain in LB media, shaking (200 rpm) under37° C. overnight (approximate 18 h), then add 30% glycerol to thefermentation broth to get final concentration of 15%, allotted to 1 mltube and kept in −20° C. as seed for production.

Add 1% of the seed to LB media, shaking (200 rpm) under 37° C. overnightto enlarge the scale of the seed, then add to RM media with a ratio of10%, culturing under 37° C. Add arabinose (20% solution) to 0.02% as aninductor when the OD600 reaches about 2.0. 4 hours after that, stop theculture process, collect the bacteria by centrifuge, resuspend thepellet with buffer A, and keep in −20° C. overnight. Thaw and break thebacteria by homogenizer, then centrifuge. Wash the pellet with buffer B,buffer C, and distilled water to get a relatively pure inclusion body.

2. Denaturation and Renaturation

Dissolve the inclusion body in Guanidine-HCl (or urea) of 6 mol/L. Thesolution will be a little cloudy. Centrifuge it at a speed of 10000 rpm.Determine the protein concentration of the supernatant. This supernatantis called “denaturation solution.” Add the denaturation solution torenaturation buffer, and keep the final protein concentration under 0.3mg/ml. It is better to add the totally denaturation solution in threesteps instead of one step. Keep the solution overnight under 4° C.Afterwards, dialyze 10 mol/L, 5 mol/L PB buffer and distilled water,then adjust its pH by 2 mol/L HAc-NaAc. Let it stand, then filtrate.

3. Purification

-   -   POROS HS/M anion exchange chromatography:

Chelating sepharose™ fast flow: Add PB buffer of 0.2 mol/L (pH 6.6) andNaCl of 4 mol/L in the solution from HS to adjust solution pH to pH 6.0and NaCl concentration to 1 mol/L.

Condense the eluted solution by POROS HS/M. Sometimes a purification bysephacryl S-100 step can be added to meet stricter purity requirements.

Note:

-   -   Buffer A: 100 mmol/L Tris-HCl, pH 7.5-10 mmol/L EDTA-100 mmol/L        NaCl    -   Buffer B: 50 mmol/L Tris-HCl, pH 7.5-1 mol/L Urea-10 mmol/L        EDTA-0.5% Triton X-100    -   Buffer C: 50 mmol/L Tris-HCl, pH 7.5-2 mol/L Urea-10 mmol/L        EDTA-0.5% Triton X-100    -   Buffer D: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 5.5)    -   Buffer E: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 5.0)    -   Buffer F: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 4.0)    -   Buffer G: 1 mol/L NaCl - - - 50 mmol/L Na₂HPO₄ (pH 3.6)    -   Renaturation buffer: 0.5 mol/L Arginine-150 mmol/L Tris-HCl, pH        7.5-0.2 mmol/L EDTA

LB Media: 1 L

Tryptone 10 g Yeast extracts  5 g NaCl 10 g

RM Media: 1 L

Casein 20 g MgCl 1 mmol/L (0.203 g) Na₂HPO₄ 4 g; KH₂PO₄ 3 g, NaCl 0.5 gNH₄Cl 1 g

After purification, the buffer was changed to PBS (pH 7.0) along withthe step of condensing by POROS HS/M. This is called the “Protein StockSolution.” It can directly used in the preparation of injections orsprays, or stored at 2-8° C.

Formula for Injection:

Solution Lyophilized powder Solution of rSIFN-co 34.5 μg/ml 34.5 μg/mlPB (pH 7.0) 25 mmol/L 10 mmol/L Glycine — 0.4 mol/L NaCl 0.1 mol/L —

For Spray:

EDTA 0.01% Tween 80 0.05% Trisodium citrate 10 mmol/L Glycerol 1.26%Sodium Chloride 0.03% Phenylmethanol  0.5% HSA  0.1% rSIFN-co 10 μg/ml

Quality Control Process

During purification, tests for protein content, protein purity, specificactivity, and pyrogen are conducted after each step. When the stocksolution is obtained, all the tests listed in the table are done oneafter the other.

The quality of the product is controlled according to “ChineseRequirements for Biologics”

1. Original Protein Solution

Lowry Item of Test Method Protein Stock Solution: Test for ProteinContent Lowry Test for Protein Purity Non-reductive SDS-PAGE (sodiumdodecyl sulfate polyacrylamide gel electrophoresis) HPLC Analysis Testfor Molecular Weights Reductive SDS-PAGE Test for Specific ActivityAccording to Method in “Specific Activity Test of Interferon Test forLeftover Exogenetic Using DNA Labeling and DNA Detection Kit Test forActivity of According to Method in Leftover Antibiotics “Chemical andOther Test Methods for Biologics” Test for Bacterial Endotoxin Accordingto Method in “Requirements for Bacterial Endotoxin Test of Biologics”Test for Isoelectronic Point Isoelectric Focusing Electrophoresis Testfor Identify UV spectrum (range of Characteristics of the wavelength:190-380 nm) Protein Peptide Mapping (hydrolyzed by pancreatic enzyme,analyzed by C-18 column) N-terminal Sequence Test C-terminal SequenceTest Circular Dichroism Amino Acid Analysis Semi-finished Product Testfor Bacterial Endotoxin According to Method in “Requirements forBacterial Endotoxin Test of Biologics” Product Appearance Check ChemicalAccording to Method in “Chemical and Other Test Methods for Biologics”Test for Specific Activity According to Method in “Specific ActivityTest of Interferon Sterility Test According to Method in “c” AbnormalToxicity Test Test on Mouse Pyrogen Test According to Method in“Requirements for Pyrogen Test of Biologics” Test for Stability ofProduct Note: “Chemical and Other Test Methods for Biologics”,“Requirements for Pyrogen Test of Biologics” and “Requirements forBacterial Endotoxin Test of Biologics” all can be found in the “ChineseRequirements for Biologics.” “Chinese Requirements for Biologics, ” PANZhengan, ZHANG Xinhui, DUAN Zhibing, et al. Chinese BiologicsStandardization committee. Published by Chemical Industry PublishingCompany, 2000.

Example 3 Stability of Lyophilized Powder of Recombinant Super-CompoundInterferon Injection

The stability experiments were carried out with samples of lyophilizedpowder of recombinant super-compound interferon (rSIFN-co) injection intwo specifications and three batches. The experiments started on April,2000.

1. Sample Source

Samples were supplied by Sichuan Huiyang Life-engineering Ltd., SichuanProvince. Lot: 990101-03, 990101-05, 990102-03, 990102-05, 990103-03,990103-05

2. Sample Specifications

Every sample in this experiment should conform with the requirements inthe table below.

TABLE 3.1 Standard of Samples in Experiment Items Standards 1.Appearance white loose powder 2. Dissolving dissolve rapidly ininjection water time (within 2 min) at room temperature 3. Claritycolorless liquid or with little milk- like glisten; should not becloudy, impurity or with indiscernible deposit 4. pH value 6.5~7.5 5.Potency (IU/dose) 80%~150% of indicated quantity (9 μg: 4.5 × 10⁶ IU, 15μg: 7.5 × 10⁶ IU) 6. Moisture no more than 3.0% (W/W)

3. Experimental Content

Test samples at 2˜8° C.: The test samples were put into a 2˜8° C.refrigerator, then the above items of these samples were respectivelytested in the 1^(st,), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th),24^(th), 30^(th), 36^(th) month. The results were recorded.

Test samples at 25° C.: The test samples were put into a thermostat at25° C., then the above items of these samples were respectively testedin the 1^(st,), 3^(rd), 6^(th), 9^(th), 12th, 18^(th), 24^(th), 30^(th)month. The results were recorded.

Test samples at 37° C.: The test samples were put into a thermostat at37° C., then the above items of these samples were respectively testedin the 1^(st,), 3^(rd), 6^(th), 9^(th), 12^(th), 18^(th), 24^(th) month.The results were recorded.

4. Results and Conclusion

-   -   1) At 37° C., according to data collected at designated points        during testing and compared with data before testing, the        potency began descending from the 6^(th) month and the changes        in the three batches were similar. The appearance of other items        had no changes.    -   2) At 25° C., according to data collected at designated points        during testing and compared with data before the testing, the        potency only had a little change, and the changes in the three        batches were similar. The appearance of other items had no        changes.    -   3). At 2-8° C., according to data collected at designated points        during testing and compared with data before testing, the        potency of the three batches all were stable. The appearance of        other items also had no changes.    -   In conclusion, it is suggested that the lyophilized powder of        recombinant super-compound interferon for injection should be        better stored and transported at low temperatures. Without such        conditions, the product can also be stored for short periods        (i.e. 3 months) at room temperature.

Example 3.5 Production Flow Chart of rSIFN-co

1. Production

1.1 Fermentation

-   -   Use mixture of LB+M9 as culturing medium. The amount of        innoculum will be 1.5%. Agitate to OD600=0.4 (about 3.5 hours)        under 32° C., then raise temperature to 42° C. Continue the        agitation for another 6 hours, the expression of rSIFN-co will        reach the maximum level. The examination under scanning of the        gel resulting from SDS-PAGE shows that the level of expression        is up to 57%, which is the highest standard in China.

1.2 Purification

The purity of the product (rSIFN-co) from this production procedure isshown to 95% under the test of SDS-PAGE where molecular weight is 14.5Kda. The reverse phase HPLC shows a single peak and the purity is up to97%. Its specific activity is up to 1×10⁹ IU/mg protein.

1.3 Packaging and Inspection

-   -   After HPLC purification, 2% human serum albumin, 1% sucrose and        1% glucose are added to the rSIFN-co. It is then separated and        lyophilized into injection sample. When tested under the        Wish-VVS inspection system, the result was 4.5×10⁸ IU. When        tested with aseptic inspection and pyrogen inspection under the        standard requirement of China, the results were negative. This        result complies with the requirements for IV injection.

2. Quality Control

2.1 Biological Characteristics

-   -   (1) When using LB+M9 to cultivate bacteria, the characteristics        should match with the typical characteristics of E-coli        bacteria. No other bacteria were detected.    -   (2) When smeared for Gram staining and inspected under a        microscope, it is bacteria-negative.    -   (3) Reaction to antibiotics is the same as those original        bacteria.    -   (4) Electron microscope inspection shows typical characteristics        of E-coli bacteria. No mycoplasma, virus spore or other micro        pollutes was detected.    -   (5) Biochemical reaction test shows characteristics of E-coli        bacteria.

2.2 Quality Control of Interferon Expression

-   -   (1) Interferon expression (cultivated in an agitating platform)        matches the amount of expression in original input bacteria.    -   (2) When tested with anti-interferon serum, a reaction is shown.    -   (3) Plasmid inspection: Restriction digest matched with the        original plasmid.

2.3 Bacteria Strain Product

-   -   Bacteria strain product denotes the specimen from the original        bacteria strain that was produced from the procedures shown in        1.2.    -   The bacteria strain product should be inspected as follows to        make sure there is no derivation: Use LB to plate 2-3 pieces and        cultivate. Separate and take 5-10 bacteria groups for the test        of interferon expression. Repeat the test at least two (2)        times. Only use the one which shows the highest % to be the        bacteria strain product.

2.4 Innoculum

-   -   The innoculum denotes the chosen bacteria strain product after        fermentation. The amount, cultivation time and most appropriate        OD value of innoculum can be decided according to bacteria        strain. An anti-polluted bacteria procedure should apply for        whatever innoculum would be produced.

2.5 Growing of Bacteria Strain

-   -   Growing of bacteria strain would be done in a Bacteria Free room        environment where no more than one bacterium is growing in the        same room. Same culturing medium will be used for both bacteria        strain and innoculum. The one used in rSIFN-co is LB.

2.6 Fermentation

-   -   (1) Fermentation only takes place in a clean fermentation room        with a single bacteria fermentation environment.    -   (2) Cleaning of fermentation container and tube is done twice,        before and after the insertion of culturing medium. Then, the        container should be frozen to reach the appropriate temperature        for innoculum.    -   (3) Avoid using antibiotic which might affect cell growth in the        culturing medium.    -   (4) Fermentation parameters like temperature, pH value,        dissolved oxygen and time required could be varied according to        different types of bacterial strains.

2.7 Bacteria Collection

-   -   (1) Centrifuge the bacteria solution to collect bacteria or use        another method. All apparatus should be cleaned before and after        the operation. The waste solution should be drained after the        cleaning procedure.    -   (2) The bacteria should be kept under 4-8° C. if they are going        to be split within 24 hours. Otherwise, they should be kept        under −30° C. Those are kept under such conditions can be used        within 6 months.

2.8 Bacteria Cell Lysis

-   -   (1) Use appropriate buffer solution to balance the bacteria        strain. Cell lysis can be done by physical, chemical or        biological methods. Use centrifuge to precipitate the bacteria        and apply cleaning solutions.    -   (2) If the chemical method is used to split cells, no solutions        harmful to human beings should be used.

2.9 Purification

-   -   (1) Purification will get rid of most of the non-interferon        contents. In the process of purification, no toxic materials        should be found if extra elements are added.    -   (2) If using antibody affinity chromatography for purification,        there should be an indication of the source and degree of        purity. Also, inspection of small quality IgG should be        performed.    -   (3) During the process of purification, clearance of pyrogen is        critical. All apparatus should be checked to eliminate this        interference.    -   (4) The highly concentrated interferon is known as “intermediate        product”. After inspection and tests, add albumin to raise the        concentration to 2% which is now known as “albumin intermediate        product”. After examination and tests, it should be kept at        −30° C. and never thawed before use. This product should be used        within 6 months.    -   (5) The albumin that is used in this process should also fulfill        tests and requirements such as: negativity under RBSAG        inspection and an indication of the ratio among monomer, dimer        and polymer.

2.10 Production into Tube Product

-   -   (1) Filtration: Use 0.22μ membrane to filter the bacteria. The        product should be handled with aseptic techniques. Samples        should be taken to test the value of the interferon.    -   (2) Dilution: Dilute the albumin intermediate product with 2%        diluent. No preservative should be added. The product can be        lyophilized after the aseptic inspection and pyrogen inspection.

2.11 Lyophilization

-   -   The lyophilization should not affect the activity of interferon,        and the water content of said lyophilite will be maintained.

2.12 Inspection

-   -   There are two types of rSIFN-co made. One is for injection and        the other for topical use. The specifications for the two are        different. There are intermediate products and final products        for each type. In the injection type, intermediate products        include purified interferon, albumin intermediate product, and        bacteria free albumin intermediate product. Final product from        the injection type will denote only lyophilized product. The        intermediate product in the topical type denotes only purified        interferon. The final product from the topical type denotes only        separated packed liquid formed lyophilized products.

2.13 Packaging

-   -   There is different packaging for the injection type and the        topical type.

2.14 Storage

-   -   The product should be kept at 4° C. The purification solution        should not be stored in a frozen state.

2.15 Expiration

-   -   The expiration period is two (2) years after the lyophilization        procedure for lyophilized products. The expiration period is 6        months after individual packing for liquidated products.

Example 4 rSIFN-co Inhibits HBV-DNA Duplication and Secretion of HBsAgand HBeAg

Materials

Solvent and Dispensing Method: Add 1 ml saline into each vial, dissolve,and mix with MEM culture medium at different concentrations. Mix on thespot.

Control drugs: IFN-α2b (Intron A) as lyophilized powder, purchased fromSchering Plough. 3×10⁶ U each, mix to 3×10⁶ IU/ml with culture medium;Infergen® (liquid solution), purchased from Amgen, 9 μg, 0.3 ml each,equal to 9×10⁶ IU, and mix with 9×10⁶ IU/ml culture medium preserve at4° C.; 2.2.15 cell: 2.2.15 cell line of hepatoma (Hep G2) cloned andtransfected by HBV DNA, constructed by Mount Sinai Medical Center.

Reagent: MEM powder, Gibco American Ltd. cattle fetal blood serum,HycloneLab American Ltd. G-418 (Geneticin); MEM dispensing, GibcoAmerican Ltd.; L-Glutamyl, imported and packaged by JING KE ChemicalLtd.; HBsAg and HBeAg solid-phase radioimmunoassay box, NorthwardReagent Institute of Chinese Isotope Ltd.; Biograncetina, Northern ChinaMedicine; And Lipofectin, Gibco American Ltd.

Experimental goods and equipment: culture bottle, Denmark Tunclon™;24-well and 96-well culture board, Corning American Ltd.; Carbon Dioxidehatching box, Shel-Lab American Ltd.; MEM culture medium 100 ml: 10%cattle fetal blood serum, 3% Glutamy 11%, G418 380 μg/ml, biograncetina50 U/ml.

Method:

2.2.15 cell culture: Added 0.25% pancreatic enzyme into culture box withfull of 2.2.15 cell, digest at 37° C. for 3 minutes, and add culturemedium to stop digest and disturb it to disperse the cells, reproducewith ratio of 1:3. They will reach full growth in 10 days.

Toxicity test: Set groups of different concentrations and a controlgroup in which cell is not acted on with medicine. Digest cell, anddispense to a 100,000 cell/ml solution. Inoculate to 96-well cultureboard, 200 μl each well, culture at 37° C. for 24 h with 5% CO₂. Testwhen simple cell layer grows.

Dispense rSIFN-co to 1.8×10⁷ IU/ml solution than prepare a series ofsolutions diluted at two-fold gradients. Add into 96-well culture board,3 wells per concentration. Change the solution every 4 days. Testcytopathic effect by microscope after 8 days. Fully destroy as 4, 75% as3, 50% as 2, 25% as 1, zero as 0. Calculate average cell lesion andinhibition rate of different concentrations. Calculate TC50 and TC0according to the Reed Muench method.

${{TC}\; 50} = {{Antilog}\left( {B + {\frac{50 + B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration,C=log dilution power

Inhibition test for HBeAg and HBsAg: Separate into positive and negativeHBeAg and HBsAg contrast groups, cell contrast group and medicineconcentration groups. Inoculate 700,000 cells/ml of 2.2.15 cell into6-well culture board, 3 ml each well, culture at 37° C. for 24 h with 5%CO₂, then prepare 5 gradiently diluted solutions with 3-fold as thegrade (Prepare 5 solutions, each with a different protein concentration.The concentration of Solution 2 is 3 times lower than that of Solution1, the concentration of Solution 3 is 3 times lower than that ofSolution 2, etc.) 4.5×10⁶ IU/ml, 1.5×10⁶ IU/ml, 0.5×10⁶ IU/ml, 0.17×10⁶¹ U/ml, and 0.056×10⁶ ¹ U/ml, 1 well per concentration, culture at 37°C. for 24 h with 5% CO₂. Change solutions every 4 days using the samesolution. Collect all culture medium on the 8^(th) day. Preserve at −20°C. Repeat test 3 times to estimate HBsAg and HBeAg with solid-phaseradioimmunoassay box (Northward Reagent Institute of Chinese IsotopeLtd.). Estimate cpm value of each well with a γ-accounting machine.

Effects calculation: Calculate cpm mean value of contrast groups anddifferent-concentration groups and their standard deviation, P/N valuesuch as inhibition rate, IC50 and SI.

-   -   1) Antigen inhibition rate

$(\%) = {\frac{A - B}{A} \times 100}$

-   -   A=cpm of control group; B=cpm of test group;    -   2) Counting the half-efficiency concentration of the medicine        -   Antigen inhibition

${{IC}\; 50} = {{Antilog}\left( {B + {\frac{50 - B}{A - B} \times C}} \right)}$

A=log>50% medicine concentration, B=log<50% medicine concentration,C=log dilution power

-   -   3) SI of interspace-conformation changed rSIFN-co effect on        HBsAg and HBeAg in 2.2.15 cell culture:

${S\; I} = \frac{{TC}\; 50}{{IC}\; 50}$

-   -   4) Estimate the differences in cpm of each dilution degree from        the control group using student t test

Southern blot: (1) HBV-DNA extract in 2.2.15 cell: Culture cell 8 days.Exsuction culture medium (Separate cells from culture medium by means ofdraining the culture medium.). Add lysis buffer to break cells, thenextract 2 times with a mixture of phenol, chloroform and isoamyl alcohol(1:1:1), 10,000 g centrifuge. Collect the supernatant adding anhydrousalcohol to deposit nucleic acid. Vacuum draw, re-dissolve into 20 μlTEbuffer. (2) Electrophoresis: Add 6×DNA loading buffer, electrophoresison 1.5% agarose gel, IV/cm, at fixed pressure for 14-18 h. (3)Denaturation and hybridization: respectively dip gel into HCl,denaturaion buffer and neutralization buffer. (4) Transmembrane: Make anorderly transfer of DNA to Hybond-N membrane. Bake, hybridize and exposewith dot blot hybridization. Scan and analyze relative density withgel-pro software. Calculate inhibition rate and IC50.

Results

Results from Tables 4.1, 4.2 and 4.3 show: After maximum innocuousconcentration exponent culturing for 8 days with 2.2.15 cell, the maximais 9.0±0×10⁶ IU/ml average inhibition rate of maximum innocuousconcentration rSIFN-co to HBeAg is 46.0±5.25% (P<O. 001), IC50 is4.54±1.32×10⁶ IU/ml, SI is 3.96; rate to HBsAg is 44.8±6.6%, IC50 is6.49±0.42×10⁶ IU/ml, SI is 2.77. This shows that rSIFN-co cansignificantly inhibit the activity of HBeAg and HBsAg, but that the IFNof the contrast group and Infergen® cannot. It has also been proved inclinic that rSIFN-co can decrease HBeAg and HBsAg or return them tonormal levels.

TABLE 4.1 Results of inhibition rate of rSIFN-co to HBsAg and HBeAgInhibition rate Average Concentration Second First Second Thirdinhibition Accumulated (×10⁴ IU/ml) First well well Third well well wellwell rate Accumulation 1-Accumulation inhibition rate First batch:(rSIFN-co) Inhibition effect to HBeAg 900 9026 8976 10476 0.4362270.43935 0.345659 0.407079 0.945909 0.592921 0.614693546 300 9616 1208210098 0.3993754 0.245347 0.369269 0.337997 0.5388299 1.2549240.300392321 100 9822 16002 12800 0.386508 0.0005 0.2005 0.1958360.200833 2.059088 0.08867188  33.33333 15770 19306 16824 0.014991 0 00.004997 0.0049969 3.054091 0.001633453  11.11111 19172 22270 18934 0 00 0 0 4.054091 0 Control Cell 16010 Blank 0 Dilution 3 IC50 602.74446016Inhibition effect to HBsAg 900 7706 7240 7114 0.342155 0.381936 0.3926930.372261 0.922258 0.627739 0.595006426 300 8856 7778 9476 0.24398160.336008 0.191053 0.257014 0.5499972 1.370724 0.286349225 100 1081810720 10330 0.07649 0.084856 0.118149 0.093165 0.292983 2.277560.113977019  33.33333 10744 11114 10570 0.082807 0.051221 0.0976610.07723 0.1998179 3.20033 0.058767408  11.11111 10672 9352 108100.088953 0.201639 0.077173 0.122588 0.122588 4.077742 0.02918541 ControlCell 11714 Blank 0 Dilution 3 IC50 641.7736749 Second batch: (rSIFN-co)Inhibition effect to HBeAg 900 7818 8516 9350 0.554378 0.514592 0.4670540.512008 1.371181 0.487992 0.737521972 300 10344 10628 9160 0.41039670.394209 0.477884 0.427497 0.8591731 1.060496 0.447563245 100 1229614228 13262 0.299134 0.18901 0.244072 0.244072 0.4316522 1.8164230.19201839  33.33333 15364 17414 16188 0.124259 0.00741 0.77291 0.0696530.1876045 2.74677 0.063933386  11.11111 17386 13632 15406 0.0090060.222982 0.121865 0.117951 0.117951 3.628819 0.03148073 Control Cell16962 Blank 0 Dilution 3 IC50 365.9357846 Inhibition effect to HBsAg 9005784 6198 5792 0.498265 0.462353 0.497571 0.486063 0.893477 0.5139370.634835847 300 7150 8534 8318 0.379771 0.259715 0.278452 0.305980.4074138 1.207957 0.252210647 100 9830 11212 10210 0.147294 0.0274120.11433 0.096345 0.101434 2.111612 0.04583464  33.33333 13942 1236813478 0 0 0 0 0.0050891 3.111612 0.001632835  11.11111 12418 11634 113520 0 0.015267 0.005089 0.005089 4.106523 0.001237728 Control Cell Blank 0Dilution 3 IC50 611.0919568 Third batch: (rSIFN-co) Inhibition effect toHBeAg 900 9702 9614 8110 0.428016 0.433204 0.521872 0.461031 1.3169830.538969 0.709599543 300 8914 10032 8870 0.4744723 0.40856 0.4770660.453366 0.8559525 1.085603 0.440859127 100 16312 12688 13934 0.0383210.251975 0.178517 0.156271 0.402586 1.929332 0.172641621  33.33333 1508012814 13288 0.110954 0.244547 0.216602 0.190701 0.2463153 2.7386310.082519158  11.11111 21928 15366 15728 0 0.094093 0.072751 0.00556150.055615 3.683017 0.014875633 Control Cell 17544 Blank 0 Dilution 3 IC50382.0496935 Inhibition effect to HBsAg 900 5616 6228 5346 0.4968640.442035 0.521054 0.486651 0.763125 0.513349 0.597838293 300 8542 85907096 0.234725 0.230425 0.364272 0.276474 0.2764738 1.236875 0.182690031100 11420 11360 11394 0 0 0 0 0 2.236875 0  33.33333 12656 11582 13110 00 0 0 0 0  11.11111 13142 12336 13342 0 0 0 0 0 4.236875 0 Control Cell11528 Blank 0 Dilution 3 IC50 694.7027149 HBeAg: Average IC50: 450.2434SD: 132.315479 HBsAg: Average IC50: 649.1894 SD: 42.29580

TABLE 4.2 Results of inhibition rate of Intron A(IFN-α2b) to HBsAg andHBeAg Con- Inhibition rate Average Accumulated centration First SecondThird First Second Third inhibition 1- inhibition (×10⁴ IU/ml) well wellwell well well well rate Accumulation Accumulation rate Inhibitioneffect to HBeAg 300 14918 11724 9950 0 0.029711 0.176529 0.0687470.068747 0.931253 0.068746724 100 14868 16890 15182 0 0 0 0 0 1.931253 0 33.33333 16760 21716 16400 0 0 0 0 0 2.931253 0  11.11111 20854 1504216168 0 0 0 0 0 3.931253 0  3.703704 12083 12083 12083 0 0 0 0 04.931253 0 Control Cell 17544 Blank 0 Dilution 3 IC50 FALSE Inhibitioneffect to HBsAg 300 9226 8196 9658 0.152489 0.247106 0.521054 0.17080.189295 0.8292 0.185857736 100 10946 10340 10828 0 0.050156 0.3642720.018495 0.0184947 1.810705 0.010110817  33.33333 12250 12980 13934 0 00 0 0 2.810705 0  11.11111 12634 12342 12000 0 0 0 0 0 3.810705 0 3.703704 10886 10886 10886 0 0 0 0 0 4.810705 0 Control Cell 10886Blank 0 Dilution 3 IC50 FALSE

TABLE 4.3 Results of inhibition rate of Infergen ® to HBsAg and HBeAgCon- Inhibition rate Average Accumulated centration First Second ThirdFirst Second Third inhibition 1- inhibition (×10⁴ IU/ml) well well wellwell well well rate Accumulation Accumulation rate First batch:(Infergen ®) Inhibition effect to HBeAg 900 14172 12156 17306 0.0916550.220869 0 0.104175 0.306157 0.895825 0.254710274 300 13390 12288 162520.1417767 0.212409 0 0.118062 0.2019827 1.777764 0.102024519 100 1436418834 14194 0.079349 0 0.090245 0.056531 0.083921 2.721232 0.02991667833.33333 15722 16034 16340 0 0 0 0 0.0273897 3.721232 0.00730659211.11111 17504 17652 14320 0 0 0.082169 0.02739 0.02739 4.6938430.005801377 Control Cell 15602 Blank 0 Dilution 3 IC50 FALSE Inhibitioneffect to HBsAg 900 12080 11692 12234 0 0.01275 0 0.00425 0.0251630.99575 0.024647111 300 12840 11484 12350 0 0.030313 0 0.0101040.0209125 1.985646 0.010422073 100 12894 14696 15086 0 0 0 0 0.0108082.985646 0.003606955  33.33333 15032 12928 13020 0 0 0 0 0.01080813.985646 0.002704416  11.11111 11794 11984 11508 0.004137 0 0.0282870.010808 0.010808 4.974837 0.002167838 Control Cell 11843 Blank 0Dilution 3 IC50 FALSE Second batch: (Infergen ®) Inhibition effect toHBeAg 900 6278 6376 6408 0.200051 0.187564 0.183486 0.190367 0.2746350.809633 0.253290505 300 7692 9092 6394 0.0198777 0 0.18527 0.0683830.0842678 1.74125 0.046161005 100 8960 7474 8190 0 0.047655 0 0.0158850.015885 2.725365 0.005794856  33.33333 8530 8144 9682 0 0 0 0 03.725365 0  11.11111 7848 7848 7848 0 0 0 0 0 4.725365 0 Control Cell7848 Blank 0 Dilution 3 IC50 FALSE Inhibition effect to HBsAg 900 1236412268 12274 0.036171 0.043655 0.043187 0.041004 0.140162 0.9589960.12751773 300 11590 12708 13716 0.0965076 0.009355 0 0.035287 0.09915811.923709 0.0490186 100 12448 13468 13982 0.029623 0 0 0.009874 0.0638712.913834 0.02144964  33.33333 12616 11346 12444 0.016526 0.1155290.029935 0.053996 0.0539965 3.859838 0.013796309  11.11111 12828 1282812828 0 0 0 0 0 4.859838 0 Control Cell 12828 Blank 0 Dilution 3 IC50FALSE Third batch: (Infergen ®) Inhibition effect to HBeAg 900 7240 66426158 0.064599 0.14186 0.204393 0.136951 0.217399 0.863049 0.201211735300 11072 8786 6902 0 0 0.108269 0.03609 0.0804479 1.82696 0.042176564100 7016 9726 7552 0.09354 0 0.024289 0.039276 0.044358 2.7876830.015663017  33.33333 7622 8866 8676 0.015245 0 0 0.005082 0.00508183.782601 0.001341671  11.11111 7740 7740 7740 0 0 0 0 0 4.782601 0Control Cell 7740 Blank 0 Dilution 3 IC50 FALSE Inhibition effect toHBsAg 900 11048 11856 11902 0.04775 0 0 0.015917 0.015917 0.9840830.015916796 300 13454 12896 11798 0 0 0 0 0 1.984083 0 100 12846 1316012546 0 0 0 0 0 2.984083 0  33.33333 12680 12458 12360 0 0 0 0 03.984083 0  11.11111 11602 11602 11602 0 0 0 0 0 4.984083 0 Control Cell11602 Blank 0 Dilution 3 IC50 FALSE HBeAg: Average IC50: 0 SD: 0 HBsAg:Average IC50: 0 SD: 0

Example 5 Preparation of rSIFN-co

Preparation of lyophilized injection Lyophilized powder Stock Solutionof 34.5 μg/ml rSIFN-co PB (pH 7.0) 10 mmol/L Glycine 0.4 mol/L

Preparation technique: Weigh materials according to recipe. Dissolvewith sterile and pyrogen-free water. Filter through 0.22 μm membrane tode-bacterialize, preserve at 6-10° C. Fill in vials after affirming itis sterile and pyrogen-free, 0.3 ml/vial or 0.5 ml/vial, and lyophilizein freeze dryer.

Preparation of liquid injection Solution Stock Solution of 34.5 μg/mlrSIFN-co PB (pH 7.0) 25 mmol/L NaCl 0.1 mol/L

Preparation: Weigh materials according to recipe. Add to desired levelwith sterile and pyrogen-free water. Filter through 0.22 μm membrane tode-bacterialize, preserve at 6-10° C. Fill in airtight vial afteraffirming it is sterile and non-pyrogen at 0.3 ml/vial or 0.5 ml/vial.Storage at 2-10° C., and protect from light.

Example 6 Acute Toxicity of rSIFN-co

Treat mice with large dose (150 μg/kg, equal to 1000 times of the normaldose per kilo used in treatment of adult patients) of rSIFN-co at onetime by intramuscular injection. Then, observe and record their deathsand toxic reactions. Results show that: 24 hours after injection, noabnormal reaction had been recorded. The organs of the animals which hadbeen selected to be killed also had no signs of abnormal changes. Thoseremaining mice were all kept alive and were normal after two weeks. Theweights of mice in the experimental group and control group allincreased, and the ratio of increase had no obvious difference betweenthe two groups (P>0.05) according to their weights on the fourteenthday. No abnormal changes were seen from the main organs of those miceafter two weeks.

1. Experimental Material

1.1 Animals

40 healthy adult mice, weighing 18-22 g, half male and half female,qualified by Sichuan experiment animal control center.

1.2 Medicines

rSIFN-co (Provided by Sichuan Huiyang Life-engineering Ltd.) sterilizedsolution, 0.15 mg/ml, Lot: 981201 rSIFN-co was administered i.m. insaline.

2. Method

Separate the 40 mice into two groups randomly, one for experimentalmedicine, another for control. Inject medicines or saline at the sameratio (0.1 ml/10 g) through muscle to each mouse according to whichgroup they belong. (150 μg/kg of rSIFN-co for experimental group; andsaline for control group). After injection, observe and record acutetoxicity shown in mice. Kill half of the mice (male and female eachhalf) to check whether there were any abnormal pathologic changes intheir main organs, such as heart, spleen, liver, lung, kidney, adrenalgland, stomach, duodenum, etc. after 24 hours. Those that remain arekept and observed until the fourteenth day. Weigh all mice, kill them,and then observe the appearance of the organs listed above to see ifthere are any abnormalities. Take pathological tissue and examine it,using the examination to assess the difference in weight increases inthe two groups.

3. Results

Results show that there was no acute toxicity seen after all mice weretreated with i.m. rSIFN-co with 150 μg/kg at a time, equal to 1000 timesthe normal dose per kilo used in treatment of adult patients. In the 14days after injection, all mice lived well. They ate, drank, exercised,and excreted normally and showed normal hair conditions. None of themdied. The observation of the main organs of the randomly selected miceshows no abnormal changes 24 hours after injection. 14 days afterinjection, all remaining mice were killed. Autopsies also showed nochanges. The weights of mice in the two groups all increased, but noobvious difference was shown when accessed with statistic method(p>0.05). See Table 6.1:

TABLE 6.1 Influence to weights of mice after injection of rSIFN-coWeights Weights Increased before after value of injection injectionweights Group Dose Animal (g) (g) (g) Control 0 20 19.8 ± 1.7 30.8 ± 2.811.0 ± 2.9 rSIFN-co 150 20 19.4 ± 1.7 32.1 ± 3.3 12.7 ± 4.3

4. Conclusion

Under conditions of this experiment, there were no toxic reactions inall mice after injection of rSIFN-co with 150 μg/kg. The conclusion canbe reached that the maximum tolerable dose of i.m. in mice is 150 μg/kg,which is equal to 1000 times the normal dose per kilo used in treatmentof adult patients.

Example 7 The Clinic Effects of Recombinant Super-Compound Interferon(rSIFN-co)

The recombinant super-compound interferon (rSIFN-co) is an invention forviral disease therapy, especially for hepatitis. Meanwhile, it caninhibit the activity of EB viruses, VSV, Herpes simplex viruses,cornaviruses, measles viruses et al. Using Wish cells/VSV system as theassay for anti-virus activity, the results showed that: the other rIFN,was 0.9×10⁸ IU/mg, Intron A was 2.0×10⁸ IU/mg and rSIFN-co was 9×10⁸IU/mg. The anti-viral activity of rSIFN-co is much higher than those ofthe former two.

Under the permission of the State Food and Drug Administration (SFDA),People's Republic of China, the clinical trials have taken place in WestChina Hospital, Sichuan University, the Second Hospital of ChongqingMedical University, the First Hospital of School of Medical, ZhejiangUniversity since the February 2003. The clinical treatment which focuseson hepatitis B is conducted under the guidance of the multicenter,double-blind random test. IFN-α1b was used as control, and the primaryresults showed the following:

The Effect of rSIFN-co Compared with IFN-α1b in the Treatment of ChronicActive Hepatitis B

1. Standard of patients selection: Standards 1-4 are effective for bothtreatment with rSIFN-co (9 μg) and IFN-α1b (5 MU, 50 μg), and Standards1-5 are for rSIFN-co (15 μg) treatment.

1). Age: 18-65

2). HBsAg test positive over last six months, HBeAg test positive, PCRassay, HBV-DNA copies ≧10⁵/ml

3). ALT≧two times the normal value

4). Never received IFN treatment; or received the Lamividine treatmentbut failed or relapsed

5). Once received other IFNs (3 MU or 5 MU) treatment six months agofollowing the standard of SFDA, but failed or relapsed

2. Evaluation of the Effects:

In reference to the recommendations from the Tenth China NationalCommittee of Virus Hepatitis and Hepatopathy, the effects were dividedinto three degrees according to the ALT level, HBV-DNA and HBeAg tests.

-   -   Response: ALT normal level, HBV-DNA negative, HBeAg negative    -   Partial response: ALT normal level, HBV-DNA or HBeAg negative    -   Non response: ALT, HBV-DNA and HBeAg unchanged    -   The response and partial response groups were considered        effective cases.

3. Results of Clinic Trial:

HBsAg HBeAg HBV-DNA Transfer Transfer Transfer Heptal to to to functionEffective negative negative negative Recover Period group Medicine casesRate rate rate rate rate  8-12 A rSIFN- 32 46.88 9.38 28.12 37.50 84.38week co(9 μg) (15)   (3)   (9)   (12)   (27)   B IFN-α1b 32 21.88 0.00 9.38 15.62 56.25 (5MU, 50 μg) (7)   (0)   (3)   (5)   (18)   16-24 ArSIFN- 64 54.69 7.81 25.00 34.38 90.62 week co(9 μg) (35)   (5)   (16)  (22)   (58)   B IFN-α1b 64 25.00 0.00  9.38 18.75 78.13 (5MU, 50 μg)(16)   (0)   (6)   (12)   (50)   Group A: treatment with rSIFN-co(9 μg)Group B: treatment with IFN-α1b (5MU, 50 μg)

In Group C, the cases were chronic active hepatitis B treatment withother IFNs (3 MU or 5 MU) before but failed or relapsed and treated withrSIFN-co (15 μg), subcutaneous injection, every one day, last 24 weeks.The total cases are 13. After 12 weeks treatment, 7 of 13 (53.85%) wereeffective. 3 of 13 (23.08%) HBeAg transferred to negative; 7 of13(53.85%) HBV-DNA transferred to negative; 11 of 13 (84.62%) hepalfunctions recovered to normal.

4. The Side Effects of rSIFN-co Compared with IFN-α1b in the Treatment

The side effects of IFN include fever, nausea, myalgia, anorexia, hairloss, leucopenia and thrombocytopenia, etc. The maximum dose of IFN-α1bis 5 MIU per time; the routine dose is 3 MIU. When taken the routinedose, 90% patients have I-II degree (WHO standard) side effects. Theyare fever lower than 38° C., nausea, myalgia, anorexia, etc. When takenat maximum dose, the rate of side effects do not rise obviously, but aremore serious. The maximum dose of rSIFN-co is 24 μg, subcutaneousinjection, every one day for 3 months. The routine dose is 9 μg. Whenroutine doses were used, less than 50% patients have I-II degree (WHOstandard) side effects, including fever below 38° C., nausea, myalgia,anorexia, leucopenia and thrombocytopenia slightly. With maximum dosage,about 50% patients suffered from leucopenia and thrombocytopenia afterusing rSIFN-co one month, but those side effects would disappear afterstopping treatment for one week. It is safe for continued use.

Observations of rSIFN-co Treatment of Hepatitis C

1. Standard of Patient Selection

-   -   1. Age: 18-65    -   2. HCV antibody positive    -   3. ALT≧1.5 times of the normal value, last more than 6 months

2. Evaluation of the Effects:

Referring to the standard of Infergen® for treatment of hepatitis C andaccording to the ALT level and HCV-RNA test, divided the effects intothree degree:

Response: ALT normal level, HCV-RNA negative

Partial response: ALT normal level, HCV-RNA unchanged

Non response: ALT and HCV-RNA unchanged.

3. Effects in Clinic

The clinical trial was done at the same time with hepatitis B treatment.46 cases received the treatment, 9 μg each time, subcutaneous injection,every day for 24 weeks. After treatment, 26 of 46 (56.52%) have obviouseffects, 12 of 46 (26.08%) HCV-RNA transferred to negative, 26 of 46(56.52%) hepal functions recovered to normal.

Example 8 Comparison of Inhibitory Effects of Different Interferons onHBV Gene Expression

Hepatitis B virus (HBV) DNA contains consensus elements fortransactivating proteins whose binding activity is regulated byinterferons. Treatment of HBV-infected hepatocytes with interferonsleads to inhibition of HBV gene expression. The aim of the present studywas to characterize the effects of different interferons on HBVregulated transcription. Using transient transfection of human hepatomacells with reporter plasmids containing the firefly luciferase geneunder the control of HBV-Enhancer (EnH) I, Enh II and core promoter,Applicant studied the biological activities of three differentinterferons on transcription.

Materials and Methods

1. Interferons: IFN-con1 (Infergen®), IFN-Hui-Yang (γSIFN-co) andIFN-beta 1b

2. Reporter plasmid: The DNA fragments containing HBV-Enhancer (EnH) I,Enh II and core promoter were prepared using PCR and blunt-end clonedinto the Smal I site of the promoter- and enhancer-less fireflyluciferase reporter plasmid pGL3-Basic (Promega, Wis., USA). Theresulting reporter plasmid was named as pGL3-HBV-Luc.

3. Cell Culture and DNA transfection: HepG2 cells were cultured in DMEMmedium supplemented with 10% FBS and 100 U/ml penicillin and 100 ug/mlstreptomycin. The cells were kept in 30° C., 5% CO2 incubator. The cellswere transfected with pGL3-HBV-Luc reporter plasmid using Boehringer'sLipofectin transfection kit. After 18 hours, the medium containingtransfection reagents was removed and fresh medium was added with orwithout interferons. The cells were kept in culture for another 48hours.

4. Luciferase Assay: Forty-eight hours after addition of interferon, thecells were harvested and cell lysis were prepared. The proteinconcentration of cell lysates were measured using Bio-Rad Protein Assaykit. The luciferase activity was measured using Promega's LuciferaseReporter Assay Systems according to the instructions of manufacturer.

Results

Expression of Luciferase Activity in Different Interferon—Treated CellLysates

No treatment IFN-con1 IFN-Hui-Yang IFN-beta 1b 100 48 + 8 29 + 6 64 + 10

This result shows that γSIFN-co inhibits most effectively on theexpression of HBV gene expression.

Example 9 Side Effects and Changes in Body Temperature when UsingγSIFN-co

There are usually more side effects to using interferon. The sideeffects includes: nausea, muscle soreness, loss of appetite, hair loss,hypoleucocytosis (hypoleukmia; hypoleukocytosis; hypoleukia), anddecrease in blood platelets, etc.

Method

Sample patients are divided into two groups. 11 patients in Group A wereinjected with 9 μg Infergen®. 10 patients in Group B were injected with9 μg γSIFN-co. Both groups were monitored for 48 hours after injections.First monitoring was recorded 1 hour after injection, after that,records were taken every 2 hours.

Table 9.1 is the comparison of side effects between patients beinginjected with 9 μg of Infergen® and 9 μg of γSIFN-co.

TABLE 9.1 Side Effects γSIFN-co Infergen ® 9 μg 9 μg Person: n = 10Person: n = 11 Body Systems Reactions Headcount Headcount In GeneralFeebleness 3 3 Sole heat 1 frigolability 3 4 Lack of 3 strength in legsMild lumbago 2 1 Body soreness 4 5 Central Nervous Headache 3 6 System/Dizziness 2 11 Peripheral Drowsiness 3 Nervous System GastroenterostomyApoclesis 1 Celiodynia 1 Diarrhea 1 Musculoskeletal Myalgia 1 2 systemArthralgia 2 Respiratory Stuffy nose 1 system Paropsia Swollen Eyes 1

Result

For those patients who were injected with γSIFN-co, the side effectswere minor. They had some common symptoms similar to flu, such as:headache, feebleness, frigolability, muscle soreness, hidrosis,arthralgia (arthrodynia; arthronalgia). The side effects of thosepatients whom were injected with Infergen® were worse than thoseinjected with γSIFN-co.

From FIGS. 8A-1, 8A-2, 8B-1, and 8B-2, it was obvious that the bodytemperatures of sample patients in Group A were higher than the patientsin Group B. It also reflected that the endurance of γSIFN-co was muchbetter than Infergen®.

Example 10 Crystal Growth of γSIFN-co and Test of CrystallographyParameter

Crystal γSIFN-co. Two types of crystal were found after systematicallytrial and experiment. (See FIGS. 9-11)

1. Crystal Growth

-   -   Dissolve γSIFN-co protein with pure water (H2O) to 3 mg/ml in        density. Search of crystallization by using Hampton Research        Crystal Screen I and II which was made by Hampton Company. By        using Drop Suspension Diffusion Method, liquid 500 μl, drop 1 μl        protein+1 μl liquid, in 293K temperature. First 2 different        types of small crystals were found as listed in Table 10.1.

TABLE 10.1 Screen of γSIFN-co Crystallin Condition I II Diluent 0.1MTris-HCl 0.1M HEPES PH = 8.75 PH = 7.13 Precipitant 17.5% (w/v) PEG550MME 10% (w/v)PEG6K Additives 0.1M NaCl 3% (v/v)MPD Temperature 293 K 293K Crystal Size (mm) 0.2 × 0.2 × 0.1 0.6 × 0.02 × 0.02 Crystallogram FIG.9 FIG. 10

2. Data Collection and Processing

-   -   Crystal I was used to collect X-Ray diffraction data and        preliminary analysis of crystallography. Parameters were also        tested. The diffraction data was collected under the room        temperature. The Crystal I (Condition I) was inserted into a        thin siliconized wall tube. Using BrukerAXS Smart CCD detector,        the light source is CuKα (λ=1.5418 Å) generated by Nonius FR591        X-ray generator. Light power 2000 KW (40 kv×50 mA), wave length        1.00 Å, under explosion 60 second, Δσ=2°, the distance between        crystal and detector was 50 mm. Data was processed using Proteum        Procedure Package by Bruker Company. See FIG. 11 for crystal        diffraction pattern (partially). See Table 10.2 for the result        of the process.

TABLE 10.2 Results of Crystallography Parameters Parameters a (Å) 82.67b (Å) 108.04 c (Å) 135.01 α (°) 90.00 β (°) 90.00 γ (°) 98.35 SpaceGroup P2 or P2₁ Sharpness of separation 5 Å Asymmetric molecule # 10Dissolution 57.6%

In addition, there was no crystal growth of γSIFN-co based on previouspublications. The closest result to the γSIFN-co was huIFN-a2b but thescreen was very complicated. After seeding 3 times, crystal grew to0.5×0.5×0.3 mm, sharpness of separation was 2.9 Å, space group was P2₁.The crystals were also big, asymmetric molecule number was 6, anddissolution was about 60%.

1. A recombinant super-compound interferon or a functional equivalentthereof with changed spatial configuration and improved efficacy.
 2. Theinterferon of claim 1, wherein the interferon is α, β, or ω. 3.(canceled)
 4. A super-compound interferon of claim 1 with uniquesecondary or tertiary structure.
 5. The super-compound interferon ofclaim 1, wherein the 3-dimensional change is the result of changes ofits production process.
 6. (canceled)
 7. (canceled)
 8. Thesuper-compound interferon of claim 5, wherein its gene is artificiallysynthesized cDNA with adjustment of its sequence from the wild-typeaccording to codon preference of E. coli.
 9. The super-compoundinterferon of claim 1, which possesses anti-viral or anti-tumoractivity.
 10. The super-compound interferon of claim 9, wherein theviral diseases comprise hepatitis A, hepatitis B, hepatitis C, othertypes of hepatitis, infections caused by Epstein-Barr virus,Cytomegalovirus, herpes simplex viruses, other herpes viruses,papovaviruses, poxviruses, picornaviruses, adenoviruses, rhinoviruses,human T-cell leukemia viruses I, human T-cell leukemia viruses II, orhuman T-cell leukemia viruses III.
 11. The super-compound interferon ofclaim 10, which directly inhibits the DNA duplication and secretion ofHBsAg and HBeAg of Hepatitis B Virus.
 12. An artificial gene which codesfor the super-compound interferon or its equivalent of claim
 1. 13. Avector comprising the gene of claim
 12. 14. An expression systemcomprising the vector of claim
 13. 15. A host cell comprising the vectorof claim
 13. 16. A process for production of recombinant super-compoundinterferon comprising introducing an artificial gene with selected codonpreference into an appropriate host, culturing said introduced host inan appropriate condition for the expression of said compound interferonand harvesting the expressed compound interferon. 17.-21. (canceled) 22.The produced super-compound interferon by the process of claim
 16. 23. Acomposition comprising the recombinant super-compound interferon ofclaim 1 and a suitable carrier.
 24. A pharmaceutical compositioncomprising the recombinant super-compound interferon of claim 1 and apharmaceutically acceptable carrier.
 25. A method for preventing ortreating viral diseases or tumors in a subject comprising administeringto the subject an effective amount of the super-compound interferon ofclaim
 1. 26. The method of claim 25 wherein the viral diseases ishepatitis A, hepatitis B, hepatitis C, other types of hepatitis,infections of viruses caused by Epstein-Barr virus, Cytomegalovirus,herpes simplex viruses, or other type of herpes viruses, papovaviruses,poxviruses, picornaviruses, adenoviruses, rhinoviruses, human T-cellleukemia viruses I, or human T-cell leukemia viruses II, or human T-cellleukemia virus III.
 27. The method of claim 25 wherein super-compoundinterferon was administered orally, via vein injection, muscleinjection, peritoneal injection, subcutaneous injection, nasal ormucosal administration, or by inhalation via an inspirator.
 28. Themethod of claim 25 wherein super-compound interferon was administeredfollowing the protocol of 9 μg or 15 μg injections per day, 3 times aweek, for a total of 24 weeks.